Whereas radiotherapy significantly prolongs the survival of patients with glioblastoma (GB), the vast majority succumb to disease within 1-2 years of diagnosis. The overall goal of this proposal is to delineate the molecules and processes that contribute to the radioresistance of GB. Traditionally, laboratory investigations of human GB radioresponse have primarily focused on tumor cell lines grown in monolayer culture and/or as leg tumor xenografts, in essence neglecting the impact of the normal brain milieu. However, our initial data indicate that intracerebral growth of established GB cell lines alters their basal and radiation-induced gene expression profiles. The overriding premise of this proposal is that the brain microenvironment has a determining impact on GB radioresponse;thus to understand the mechanisms mediating GB radioresistance it will be necessary to account for its unique in situ circumstances. Towards this end, the proposed studies will focus on intracerebral xenografts grown from CD133+ GB tumor initiating cells (TICs). In contrast to established cell lines, intracerebral xenografts grown from TICs replicate the genotype/phenotype and in vivo growth pattern of primary GBs;such xenografts are then also likely to best simulate the consequences of the brain microenvironment on GB radioresponse. The proposed studies will involve 3 Aims. The first aim will define the influence of orthotopic growth on the basal and radiation-induced gene expression profiles of GB TICs. These studies will involve microarray-based transcriptome and gene translation analyses of GB TICs grown in vitro and in vivo as leg tumor and intracerebral xenografts. These genome-wide interrogations will test the hypothesis that the orthotopic environment uniquely influences GB TIC gene expression. Along these lines, an additional hypothesis to be tested is that the radioresponse of GB TICs includes proteins uniquely expressed or induced under intracerebral conditions. The second aim will determine whether the radioresponse of cells within a TIC intracerebral xenograft differ according to topology and/or phenotype. These studies will test the hypothesis that the intra-tumor heterogeneity generated by orthotopic growth results in subpopulations with varying degrees of radiosensitivity. Finally, the third aim is to test the hypothesis that there are targets for GB radiosensitization unique to the orthotopic environment. Studies will initially define the ability of single and fractionated radiation protocols to control the growth of TIC intracerebral xenograft. Targeted strategies suggested from Aims 1 and 2 will then be tested for their ability to enhance radiation-induced growth control. It is anticipated that these studies will generate novel insights into the molecular and cellular determinants of GB radioresistance and thus provide the basis for developing novel strategies for their radiosensitization. PUBLIC HEALTH RELEVANCE: Improving the efficacy of radiation as a treatment for glioblastoma (GB) will require a thorough understanding of the molecules and processes that contribute to their radioresistance, which necessitates taking into account the impact of the microenvironment. Towards this end, the proposed studies will delineate the fundamental determinants of GB radioresponse using orthotopic xenograft models. It is anticipated that these studies will provide novel insight into GB radioresistance and thus provide the basis for developing novel strategies for their radiosensitization.